Method of using anti-IL-13 antibodies

ABSTRACT

The present invention relates to human anti-IL-13 binding molecules, particularly antibodies, and to methods for using anti-IL-13 antibody molecules in diagnosis or treatment of IL-13 related disorders, such as asthma, atopic dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease and Hodgkin&#39;s lymphoma.

This application is a Divisional of U.S. application Ser. No. 12/091,020filed 22 Apr. 2008 now U.S. Pat. No. 7,910,708 which is a U.S. NationalPhase filing of PCT/EP2006/010098 filed 19 Oct. 2006, which claimspriority to GB Patent Application 0616666.4 filed 22 Aug. 2006, and GBPatent Application 0521509.0 filed 21 Oct. 2005, the contents of each ofwhich are incorporated herein by reference in their entirety.

FIELD OF USE

The present invention relates to specific binding members, in particularhuman anti-IL-13 antibody molecules and especially those whichneutralize IL-13 activity. It further relates to methods for usinganti-IL-13 antibody molecules in diagnosis or treatment of IL-13 relateddisorders, such as asthma, atopic dermatitis, allergic rhinitis,fibrosis, inflammatory bowel disease and Hodgkin's lymphoma.

BACKGROUND OF THE INVENTION

Interleukin (IL)-13 is a 114 amino acid cytokine with an unmodifiedmolecular mass of approximately 12 kDa [McKenzie, A. N., et al. JImmunol, 1993. 150 (12): p. 5436-44, and Minty, A., et al. Nature, 1993.362 (6417): p. 248-50.]. IL-13 is most closely related to IL-4 withwhich it shares 30% sequence similarity at the amino acid level. Thehuman IL-13 gene is located on chromosome 5q31 adjacent to the IL-4gene. This region of chromosome 5q contains gene sequences for other Th2lymphocyte derived cytokines including GM-CSF and IL-5, whose levelstogether with IL-4 have been shown to correlate with disease severity inasthmatics and rodent models of allergic inflammation [Nakamura, Y., etal. Am J Respir Cell Mol Biol, 1996. 15 (5): p. 680-7, Robinson, D. S.,et al. N Engl J Med, 1992. 326 (5): p. 298-304, Walker, C., et al. Am JRespir Crit Care Med, 1994. 150 (4): p. 1038-48, Humbert, M., et al. AmJ Respir Crit Care Med, 1996, 154 (5): p. 1497-504, Corrigan, C. J. andA. B. Kay Int Arch Allergy Appl Immunol, 1991. 94 (1-4): p. 270-1,Bentley, A. M., et al. Am J Respir Cell Mol Biol, 1993.].

Although initially identified as a Th2 CD4+ lymphocyte derived cytokine,IL-13 is also produced by Th1 CD4+ T-cells, CD8+ T lymphocytes NK cells,and non-T-cell populations such as mast cells, basophils, eosinophils,macrophages, monocytes and airway smooth muscle cells.

IL-13 is reported to mediate its effects through a receptor system thatincludes the IL-4 receptor a chain (IL-4Rα)-, which itself can bind IL-4but not IL-13, and at least two other cell surface proteins, IL-13Rα₁and IL-13Rα₂ [Murata, T., et al. Int J Hematol, 1999. 69(1): p. 13-20,Andrews, A. L., et al. J Biol Chem, 2002. 277(48): p. 46073-8.].IL-13Rα₁ can bind IL-13 with low affinity, subsequently recruitingIL-4Rα to form a high affinity functional receptor that signals [Miloux,B., et al. FEBS Lett, 1997. 401 (2-3): p. 163-6, Hilton, D. J., et al.Proc Natl Acad Sci USA, 1996. 93 (1): p. 497-501]. The Genbank databaselists the amino acid sequence and the nucleic acid sequence of IL-13Rα₁as NP 001551 and Y10659 respectively. Studies in STAT6 (signaltransducer and activator of transcription 6)-deficient mice haverevealed that IL-13, in a manner similar to IL-4, signals by utilizingthe JAK-STAT6 pathway [Kuperman, D., et al. J Exp Med, 1998. 187 (6): p.939-48, Nelms, K., et al. Annu Rev Immunol, 1999. 17: p. 701-38.].IL-13Rα₂ shares 37% sequence identity with IL-13Rα₁ at the amino acidlevel and binds IL-13 with high affinity [Zhang, J. G., et al. J BiolChem, 1997. 272 (14): p. 9474-80, Caput, D., et al. J Biol Chem, 1996.271 (28): p. 16921-6.]. However, IL-13Rα₂ has a shorter cytoplasmic tailthat lacks known signaling motifs. Cells expressing IL-13Rα₂ are notresponsive to IL-13 even in the presence of IL-4Rα [Kawakami, K., et al.Blood, 2001. 97 (9): p. 2673-9]. It is postulated, therefore, thatIL-13Rα₂ acts as a decoy receptor regulating IL-13 but not IL-4function. This is supported by studies in IL-13Rα₂ deficient mice whosephenotype was consistent with increased responsiveness to IL-13 [Wood,N., et al. J Exp Med, 2003. 197 (6): p. 703-709, Chiaramonte, M. G., etal. J Exp Med, 2003. 197 (6): p. 687-701]. The Genbank database liststhe amino acid sequence and the nucleic acid sequence of IL-13Rα₂ asNP000631 and Y08768 respectively.

SUMMARY OF THE INVENTION

An embodiment of the invention herein provides an isolated human orhumanized antibody or functional fragment thereof with anantigen-binding region that is specific for target protein IL-13 and theantibody or functional fragment thereof binds to IL-13. In a relatedembodiment, the binding to IL-13 is determined at least by cell surfaceIL-13 receptor binding preventing inflammatory mediator release.

In still another embodiment, the invention provides an isolatedantigen-binding region of an antibody or functional fragment thereof Incertain embodiments, the isolated antigen-binding region includes anH-CDR3 region having an amino acid sequence selected from SEQ ID NOs:9-10, and conservative variants thereof. As described herein, theconservative variants include amino acid residues in any of the aminoacid sequences identified. In a related embodiment, the isolatedantigen-binding region is an H-CDR2 region having the amino acidsequence of SEQ ID NO: 8, and conservative variants thereof. In anotherrelated embodiment, the isolated antigen-binding region is an H-CDR1region having an amino acid sequence selected from SEQ ID NO: 6-7, andconservative variants thereof.

In another embodiment, the isolated antigen-binding region is an L-CDR3region having an amino acid sequence selected from SEQ ID NOs: 20-22,and conservative variants thereof. In still another related embodiment,the isolated antigen-binding region is an L-CDR1 region having an aminoacid sequence selected from SEQ ID NOs: 16-18, and conservative variantsthereof. In yet another related embodiment, the isolated antigen-bindingregion is an L-CDR2 region having the amino acid sequence of SEQ ID NO:19, and conservative variants thereof.

In certain embodiments, the isolated antigen-binding region is avariable light chain having an amino acid sequence selected from SEQ ID16-22, and conservative variants thereof.

In another embodiment, the isolated antigen-binding region is a heavychain having an amino acid sequence selected from one to three of SEQ ID6-10, and a sequence having at least 60, 70, 80, 90 or 95 percentsequence identity in the CDR regions with the CDR regions having SEQ IDNOs: 6-10. In a related embodiment, the isolated antigen-binding regionis a light chain having an amino acid sequence selected from one tothree of SEQ ID NOs: 16-22, and a sequence having at least 60, 70, 80,90 or 95 percent sequence identity in the CDR regions with the CDRregions having SEQ ID NOs: 16-22.

In a certain embodiment, the isolated antibody is an IgG. In anotherembodiment, the isolated antibody is an IgG1 or an IgG4.

In yet another embodiment, the invention provides an isolated human orhumanized antibody or functional fragment thereof, having anantigen-binding region that is specific for an epitope of IL-13, and theantibody or functional fragment binds to IL-13 surface receptors on acell. In a related embodiment, the invention provides an isolated humanor humanized antibody or functional fragment thereof, having anantigen-binding region that is specific for an epitope of target IL-13,and the epitope contains one or more amino acid residues of amino acidresidues 1-112 of target IL-13. In a related embodiment, the epitope isa conformational epitope.

In yet another embodiment, the antibody or functional fragment is a Fabor scFv antibody fragment. In a related embodiment, the isolatedantibody is an IgG. In another related embodiment, the isolated antibodyis an IgG1 or an IgG4.

In another embodiment, the invention provides a pharmaceuticalcomposition having at least one of any of the above antibodies orfunctional fragments or conservative variants, and a pharmaceuticallyacceptable carrier or excipient therefor.

In still another embodiment, the invention provides for a transgenicanimal carrying a gene encoding any of the above antibodies orfunctional fragments thereof.

In certain embodiments, the invention provides a method for treating adisorder or condition associated with the presence of a cell having areceptor target for IL-13. The method involves administering to asubject in need thereof an effective amount of any of the abovepharmaceutical compositions. In a related embodiment, the disorder orcondition to be treated is a respiratory disorder.

In another embodiment, the disorder or condition to be treated isbronchial asthma, which is a common persistent inflammatory disease ofthe lung characterised by airways hyper-responsiveness (AHR), mucusoverproduction, fibrosis and raised serum IgE levels. Li et al, Abstractfor poster submitted at The American Thoraics Society Annual Meeting,2003, Seattle, reported affects of a neutralising anti-mouse IL-13antibody in a chronic mouse model of asthma.

In another embodiment, the disorder or condition to be treated isChronic Obstructive Pulmonary Disease (COPD). Zheng et al J Clin Invest,2000. 106 (9): p. 1081-93, have demonstrated that over expression ofIL-13 in the mouse lung caused emphysema, elevated mucus production andinflammation, reflecting aspects of human COPD. mRNA levels of IL-13have been shown to be higher in autopsy tissue samples from subjectswith a history of COPD when compared to lung samples from subjects withno reported lung disease (J. Elias, Oral communication at AmericanThoracic Society Annual Meeting 2002). In another study, raised levelsof IL-13 were demonstrated by immunohistochemistry in peripheral lungsections from COPD patients [Wardlaw, A. J., Clin Med, 2001. 1 (3): p.214-8.].

In another embodiment, the disorder or condition to be treated isselected from other inflammatory or obstructive airways diseases andconditions such as acute lung injury (ALI), acute/adult respiratorydistress syndrome (ARDS), dyspnea, allergic airway inflammation, smallairway disease, lung carcinoma, acute chest syndrome in patients withsickle cell disease and pulmonary hypertension, as well as exacerbationof airways hyperreactivity consequent to other drug therapy, inparticular other inhaled drug therapy.

In another embodiment, the disorder or condition to be treated isbronchitis of whatever type or genesis including, e.g., acute,arachidic, catarrhal, croupus, chronic or phthinoid bronchitis.

In another embodiment, the disorder or condition to be treated includespneumoconiosis (an inflammatory, commonly occupational, disease of thelungs, frequently accompanied by airways obstruction, whether chronic oracute, and occasioned by repeated inhalation of dusts) of whatever typeor genesis, including, for example, aluminosis, anthracosis, asbestosis,chalicosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis.

In another embodiment, the disorder or condition to be treated isselected from atopic rhinitis (hay fever), allergic dermatitis (eczema)and chronic sinusitis. Raised levels of IL-13 have been measured inhuman subjects with atopic rhinitis (hay fever), allergic dermatitis(eczema) and chronic sinusitis. For example levels of IL-13 were foundto be higher in bronchial biopsies, sputum and broncho-alveolar lavage(BAL) cells from asthmatics compared to control subjects [Humbert, M.,et al. J Allergy Clin Immunol, 1997. 99 (5): p. 657-65, Kotsimbos, T.C., P. Ernst, and Q. A. Hamid, Proc Assoc Am Physicians, 1996. 108 (5):p. 368-73, Komai-Koma, M., F. Y. Liew, and P. C. Wilkinson, J Immunol,1995. 155 (3): p. 1110-6, Naseer, T., et al. Am J Respir Crit Care Med,1997].

In another embodiment, the disorder or condition to be treated isselected from other inflammatory conditions of the skin, for example,psoriasis or lupus erythematosus.

In another embodiment, the disorder or condition to be treated isinflammatory bowel disease, such as ulcerative colitis and Crohn'sdisease. Heller et al. (2002) Immunity, 17 (5): 629-38, report thatneutralisation of IL-13 by administration of soluble IL-13Ra2ameliorated colonic inflammation in a murine model of human ulcerativecolitis. Correspondingly, IL-13 expression was higher in rectal biopsyspecimens from ulcerative colitis patients when compared to controls.

In another embodiment, the disorder or condition to be treated isselected from other fibrotic conditions, such as systemic sclerosis,pulmonary fibrosis, idiopathic pulmonary fibrosis or fibroid lung.Increased levels of IL-13 have been measured in the serum of patientswith systemic sclerosis [Hasegawa, M., et al. J Rheurnatol, 1997. 24(2): p. 328-32] and in BAL samples from patients affected with otherforms of pulmonary fibrosis [Hancock, A., et al. Am J Respir Cell MolBiol, 1998].

In another embodiment, the disorder or condition to be treated is liverfibrosis. Specific inhibition of IL-13 by administration of solubleIL-13Ra2 or IL-13 gene disruption, but not ablation of IL-4 productionprevented fibrogenesis in the liver [Fallon, P. G., et al. J Immunol,2000. 164 (5): p. 2585-91, Chiaramonte, M. G., et al. J Clin Invest,1999. 104 (6): p. 777-85, Chiaramonte, M. G., et al. Hepatology, 2001.34(2): p. 273-82.].

In another embodiment, the disorder or condition to be treated isHodgkin's disease. Hodgkin's disease is unusual among malignancies inthat the neoplastic Reed-Sternberg cell, often derived from B-cells,make up only a small proportion of the clinically detectable mass.Hodgkin's disease-derived cell lines and primary ReedSternberg cellsfrequently express IL-13 and its receptor [Skinnider, a F., et al.Blood, 2001. 97(1): p. 250-5]. As IL-13 promotes cell survival andproliferation in normal B-cells, it was proposed that IL-13 could act asa growth factor for Reed-Sternberg cells. Skinnider et al. havedemonstrated that neutralising antibodies against IL-13 can inhibit thegrowth of Hodgkin's disease-derived cell lines in vitro [Kapp, U., etal. J Exp Med, 1999. 189 (12): p. 1939-46.]. This finding suggested thatReed-Sternberg cells might enhance their own survival by an IL-13autocrine and paracrine cytokine loop. Consistent with this hypothesis,raised levels of IL-13 have been detected in the serum of some Hodgkin'sdisease patients when compared to normal controls [Fiumara, P., F.Cabanillas, and A. Younes, Blood, 2001. 98 (9): p.2877-8.]. IL-13inhibitors may therefore prevent disease progression by inhibitingproliferation of malignant Reed-Sternberg cells.

In another embodiment, the disorder or condition to be treated is tumourrecurrence or metastasis. Inhibition of IL-13 has been shown to enhanceanti-viral vaccines in animal models and may be beneficial in thetreatment of HIV and other infectious diseases [Ahlers, J. D., et al.Proc Natl Acad Sci USA, 2002]. Many human cancer cells expressimmunogenic tumour specific antigens. However, although many tumoursspontaneously regress, a number evade the immune system(immunosurveillance) by suppressing T-cell mediated immunity. Terabe etal. Nat Immunol, 2000. 1 (6): p. 515-20, have demonstrated a role ofIL-13 in immunosuppression in a mouse model in which tumoursspontaneously regress after initial growth and then recur. Specificinhibition of IL-13, with soluble IL-13Ra2, protected these mice fromtumour recurrence. Terabe et al went on to show that IL-13 suppressesthe differentiation of tumour specific CD8+ cytotoxic lymphocytes thatmediate anti-tumour immune responses.

In another embodiment, the disorder or condition to be treated is arespiratory viral infection, which exacerbates underlying chronicconditions such as asthma, chronic bronchitis, COPD, otitis media, andsinusitis. The respiratory viral infection treated may be associatedwith secondary bacterial infection, such as otitis media, sinusitis orpneumonia.

In another embodiment, the disorder or condition to be treated isselected from other diseases or conditions, in particular diseases orconditions having an inflammatory component, for example, diseases ofthe bone and joints including rheumatoid arthritis, psoriatic arthritis,and other diseases such as atherosclerosis, multiple sclerosis, andacute and chronic allograft rejection, e.g. following transplantation ofheart, kidney, liver, lung or bone marrow.

In another embodiment, the disorder or condition to be treated isendotoxic shock, glomerulonephritis, cerebral and cardiac ischemia,Alzheimer's disease, cystic fibrosis, virus infections and theexacerbations associated with them, acquired immune deficiency syndrome(AIDS), multiple sclerosis (MS), Helicobacter pylori associatedgastritis, and cancers, particularly the growth of ovarian cancer.

In another embodiment, the disorder or condition to be treated is thesymptoms caused by viral infection in a human which is caused by thehuman rhinovirus, other enterovirus, coronavirus, herpes viruses,influenza virus, parainfluenza virus, respiratory syncytial virus or anadenovirus.

Treatment in accordance with the present invention may be symptomatic orprophylactic.

The effectiveness of an agent of the invention in inhibitinginflammatory conditions, for example in inflammatory airways diseases,may be demonstrated in an animal model, e.g. mouse, rat or rabbit model,of airway inflammation or other inflammatory conditions, for example asdescribed by Wada et al, J. Exp. Med (1994) 180:1135-40; Sekido et al,Nature (1993) 365:654-57; Modelska et al., Am. J. Respir. Crit. Care.Med (1999) 160:1450-56; and Laffon et al (1999) Am. J. Respir. Crit.Care Med. 160:1443-49.

In yet another embodiment, the invention provides a method foridentifying a cell having a receptor for IL-13. This method involvescontacting the cell with any of the above antibodies or antibodyfragments further having a detectable label. The label is radioactive,fluorescent, magnetic, paramagnetic, or chemiluminescent. The methodfurther can involve any of the above imaging or separating the labeledcell.

In another embodiment, any of the above human or humanized antibodies orantibody fragments are synthetic.

In another embodiment, the invention provides a pharmaceuticalcomposition and an additional therapeutic agent.

The additional therapeutic agent can be selected from the groupconsisting of anti-inflammatory, bronchodilatory, antihistamine oranti-tussive drug substances, particularly in the treatment ofobstructive or inflammatory airways diseases such as those mentionedhereinbefore, for example as potentiators of therapeutic activity ofsuch drugs or as a means of reducing required dosaging or potential sideeffects of such drugs. A therapeutic agent of the invention may be mixedwith the other drug substance in a fixed pharmaceutical composition orit may be administered separately, before, simultaneously with or afterthe other drug substance. Accordingly the invention includes acombination of an agent of the invention as hereinbefore described withan anti-inflammatory, bronchodilatory, antihistamine or anti-tussivedrug substance, said agent of the invention and said drug substancebeing in the same or different pharmaceutical composition.

Suitable anti-inflammatory drugs include steroids, in particularglucocorticosteroids such as budesonide, beclamethasone dipropionate,fluticasone propionate, ciclesonide or mometasone furoate, or steroidsdescribed in WO 02/88167, WO 02/12266, WO 02/100879, WO 02/00679(especially those of Examples 3, 11, 14, 17, 19, 26, 34, 37, 39, 51, 60,67, 72, 73, 90, 99 and 101), WO 03/35668, WO 03/48181, WO 03/62259, WO03/64445, WO 03/72592, WO 04/39827 and WO 04/66920; non-steroidalglucocorticoid receptor agonists, such as those described in DE10261874, WO 00/00531, WO 02/10143, WO 03/82280, WO 03/82787, WO03/86294, WO 03/104195, WO 03/101932, WO 04/05229, WO 04/18429, WO04/19935 and WO 04/26248; LTB4 antagonists such as BIIL 284, CP-195543,DPC11870, LTB4 ethanolamide, LY 293111, LY 255283, CGS025019C,CP-195543, ONO-4057, SB 209247, SC-53228 and those described in U.S.Pat. No. 5,451,700; LTD4 antagonists such include montelukast,pranlukast, zafirlukast, accolate, SR2640, Wy-48,252, ICI 198615,MK-571, LY-171883, Ro 24-5913 and L-648051; PDE4 inhibitors suchcilomilast (Ariflo® GlaxoSmithKline), Roflumilast (Byk Gulden), V-11294A(Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plough), Arofylline(Almirall Prodesfarma), PD189659/PD168787 (Parke-Davis), AWD-12-281(Asta Medica), CDC-801 (Celgene), SelCID™ CC-10004 (Celgene),VM554/UM565 (Vernalis), T-440 (Tanabe), KW-4490 (Kyowa Hakko Kogyo), andthose disclosed in WO 92/19594, WO 93/19749, WO 93/19750, WO 93/19751,WO 98/18796, WO 99/16766, WO 01/13953, WO 03/104204, WO 03/104205, WO03/39544, WO 04/000814, WO 04/000839, WO 04/005258, WO 04/018450, WO04/018451, WO 04/018457, WO 04/018465, WO 04/018431, WO 04/018449, WO04/018450, WO 04/018451, WO 04/018457, WO 04/018465, WO 04/019944, WO04/019945, WO 04/045607 and WO 04/037805; A_(2A) agonists such as thosedescribed in EP 1052264, EP 1241176, EP 409595A2, WO 94/17090, WO96/02543, WO 96/02553, WO 98/28319, WO 99/24449, WO 99/24450, WO99/24451, WO 99/38877, WO 99/41267, WO 99/67263, WO 99/67264, WO99/67265, WO 99/67266, WO 00/23457, WO 00/77018, WO 00/78774, WO01/23399, WO 01/27130, WO 01/27131, WO 01/60835, WO 01/94368, WO02/00676, WO 02/22630, WO 02/96462, and WO 03/086408; and A_(2B)antagonists such as those described in WO 02/42298.

Suitable bronchodilatory drugs include anticholinergic or antimuscarinicagents, in particular ipratropium bromide, oxitropium bromide,tiotropium salts and CHF 4226 (Chiesi), and glycopyrrolate, but alsothose described in EP 424021, U.S. Pat. No. 3,714,357, U.S. Pat. No.5,171,744, WO 01/04118, WO 02/00652, WO 02/51841, WO 02/53564, WO03/00840, WO 03/33495, WO 03/53966, WO 03/87094, WO 04/018422 and WO04/05285; and beta-2 adrenoceptor agonists such as albuterol(salbutamol), metaproterenol, terbutaline, salmeterol fenoterol,procaterol, and especially, formoterol, carmoterol and pharmaceuticallyacceptable salts thereof, and compounds (in free or salt or solvateform) of formula 1 of WO 00/75114, which document is incorporated hereinby reference, preferably compounds of the Examples thereof, especially acompound of formula

i.e.,(5-[(R)-2-(5,6-Diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one)and pharmaceutically acceptable salts thereof, as well as compounds (infree or salt or solvate form) of formula I of WO 04/16601, and alsocompounds of EP 1440966, JP 05025045, WO 93/18007, WO 99/64035, US2002/0055651, WO 01/42193, WO 01/83462, WO 02/66422, WO 02/70490, WO02/76933, WO 03124439, WO 03/42160, WO 03/42164, WO 03/72539, WO03/91204, WO 03/99764, WO 04/16578, WO 04122547, WO 04/32921, WO04/33412, WO 04/37768, WO 04/37773, WO 04/37807, WO 04/39762, WO04/39766, WO 04/45618 WO 04/46083, WO 04/80964, EP1460064, WO 04/087142,WO 04/089892, EP 01477167, US 2004/0242622, US 2004/0229904, WO04/108675, WO 04/108676, WO 05/033121, WO 05/040103 and WO 05/044787.

Suitable dual anti-inflammatory and bronchodilatory drugs include dualbeta-2 adrenoceptor agonist/muscarinic antagonists such as thosedisclosed in US 2004/0167167, WO 04/74246 and WO 04/74812.

Suitable antihistamine drug substances include cetirizine hydrochloride,acetaminophen, clemastine fumarate, promethazine, loratidine,desloratidine, diphenhydramine and fexofenadine hydrochloride,activastine, astemizole, azelastine, ebastine, epinastine, mizolastineand tefenadine as well as those disclosed in JP 2004107299, WO 03/099807and WO 04/026841.

Combinations of therapeutic agents of the invention and anticholinergicor antimuscarinic agents, steroids, beta-2 agonists, PDE4 inhibitors,dopamine receptor agonists, LTD4 antagonists or LTB4 antagonists mayalso be used. Other useful combinations of agents of the invention withanti-inflammatory drugs are those with other antagonists of chemokinereceptors, e.g. CCR-1, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5antagonists such as Schering-Plough antagonists SC-351125, SCH-55700 andSCH-D, Takeda antagonists such asN-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H-benzocyclohepten-8-yl]carbonyl]amino]phenyl]-methyl]-tetrahydro-N,N-dimethyl-2H-pyran-4-aminiumchloride (TAK-770), CCR-5 antagonists described in U.S. Pat. No.6,166,037 (particularly claims 18 and 19), WO 0066558 (particularlyclaim 8), WO 0066559 (particularly claim 9), WO 04/018425 and WO04/026873.

The additional therapeutic agent may also be selected from the groupconsisting of other cytokine binding molecules, particularly antibodiesof other cytokines, in particular a combination with an anti-IL4antibody, such as described in PCT/EP2005/00836, an anti-IgE antibody,such as Xolair®, an anti-IL31 antibody, an anti-IL31R antibody, ananti-TSLP antibody, an anti-TSLP receptor antibody, an anti-endoglinantibody, an anti-IL1b antibody or another anti-IL13 antibody, such asdescribed in WO05/007699.

In a certain embodiment, the invention provides an antibody having afirst amino acid sequence which is a heavy chain selected from one tothree of SEQ ID NOs: 6-10, and a sequence having at least 60, 70, 80, 90or 95 percent sequence identity in the CDR regions with the CDR regionshaving SEQ ID NOs: 6-10; and a second amino acid sequence which is alight chain selected from one to three of SEQ ID NOs: 16-22, and asequence having at least 60, 70, 80, 90 or 95 percent sequence identityin the CDR regions with the CDR regions shown in SEQ ID NOs: 16-22.

In still another embodiment, the invention provides an immunoconjugatemade out of a first component which is an antibody or fragment thereofand a second component having a second amino acid sequence. For example,the immunoconjugate is a cytotoxin, or the immunoconjugate is a bindingprotein or antibody having a binding specificity for a target that isdifferent from IL-13.

In certain embodiments, the invention provides for a bispecificantibody.

In another embodiment, the invention provides a kit having an antibodyor antibody fragment thereof. In some embodiments, the kit furthercontains a pharmaceutically acceptable carrier or excipient therefore.In other related embodiments, the antibody in the kit is present in aunit dose. In yet another related embodiment, the kit includesinstructions for use in administering to a subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated antibodies, particularly humanantibodies, that bind specifically to IL-13 and that inhibit functionalproperties of IL-13. In certain embodiments, the antibodies of theinvention are derived from particular heavy and light chain sequencesand/or comprise particular structural features such as CDR regionscomprising particular amino acid sequences. The invention providesisolated antibodies, methods of making such antibodies, immunoconjugatesand bispecific molecules comprising such antibodies and pharmaceuticalcompositions containing the antibodies, immunconjugates or bispecificmolecules of the invention. The invention also relates to methods ofusing the antibodies to inhibit a disorder or condition associated withthe presence of cell receptor target IL-13, for example, in thetreatment of an inflammatory or allergic condition, particularly aninflammatory or obstructive airways disease.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term ‘interleukin-13’ or ‘IL-13’ is, except where context dictatesotherwise, reference to human IL-13. The present invention providesantibodies to human IL-13, especially human antibodies, that arecross-reactive with non-human primate IL-13, including cynomolgus andrhesus monkey IL-13. Antibodies in accordance with some embodiments ofthe present invention recognise a variant of IL-13 in which the arginineresidue at amino acid position 130 is replaced by glutamine. In otheraspects and embodiments the present invention provides specific bindingmembers against murine IL-13, specifically mouse IL-13.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and capable of the transmission of such a signalacross the plasma membrane of a cell. An example of a “cell surfacereceptor” of the present invention is the IL-13 receptor to which theIL-13 protein molecule binds.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i. e., “antigen-binding portion”) orsingle chains thereof. A naturally occurring “antibody” is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as V_(H)) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as V_(L))and a light chain constant region. The light chain constant region iscomprised of one domain, C_(L). The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is, composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., IL-13). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and CH1domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds IL-13is substantially free of antibodies that specifically bind antigensother than IL-13). An isolated antibody that specifically binds IL-13may, however, have cross-reactivity to other antigens, such as IL-13molecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies of theinvention may include amino acid residues not encoded by human sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo). However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgE,IgG such as IgG1 or IgG4) that is encoded by the heavy chain constantregion genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

As used herein, an antibody that “specifically binds to human IL-13” isintended to refer to an antibody that binds to human IL-13 with a K_(D)of 5×10⁻⁹ M or less. An antibody that “cross-reacts with an antigenother than human IL-13” is intended to refer to an antibody that bindsthat antigen with a 5×10⁻⁹ M or less. An antibody that “does notcross-react with a particular antigen” is intended to refer to anantibody that binds to that antigen, with a K_(D) of 1.5×10⁻⁸ M orgreater, or a K_(D) of 5-10×10⁻⁸ M or 1×10⁻⁷ M or greater. In certainembodiments, such antibodies that do not cross-react with the antigenexhibit essentially undetectable binding against these proteins instandard binding assays.

As used herein, an antibody that “inhibits binding of IL-13 to the IL-13receptor” refers to an antibody that inhibits IL-13 binding to thereceptor with a KD of 5 nM or less.

As used herein, an antibody that “inhibits inflammatory mediatorrelease” is intended to refer to an antibody that inhibits IL-13 inducedeotaxin release from human lung fibroblasts with an IC₅₀ less than 10nM, 5 nM, 2.5 nM, 1.0 nM, 0.5 nM, or less.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(D),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, 10⁻⁹ M or less, or 10⁻¹⁰ Mor less for a target antigen.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

Standard assays to evaluate the binding ability of the antibodies towardIL-13 of various species are known in the art, including for example,ELISAs, western blots and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis. Assays to evaluate the effects of theantibodies on functional properties of IL-13 are described in furtherdetail in the Examples.

Accordingly, an antibody that “inhibits” one or more of these IL-13functional properties (e.g., biochemical, immunochemical, cellular,physiological or other biological activities, or the like) as determinedaccording to methodologies known to the art and described herein, willbe understood to relate to a statistically significant decrease in theparticular activity relative to that seen in the absence of the antibody(e.g., or when a control antibody of irrelevant specificity is present).An antibody that inhibits IL-13 activity effects such a statisticallysignificant decrease by at least 10% of the measured parameter, by atleast 50%, 80% or 90%, and in certain embodiments an antibody of theinvention may inhibit greater than 95%, 98% or 99% of IL-13 functionalactivity.

Monoclonal Antibodies

Antibodies of the invention are the human monoclonal antibodies,isolated and structurally characterized as described, in Examples 1-5.The V_(H) amino acid sequences of the antibodies are shown in SEQ IDNOs: 6-10 respectively. The V_(L) amino acid sequences of the antibodiesare shown in SEQ ID NOs: 16-22 respectively. Other antibodies of theinvention include amino acids that have been mutated, yet have at least60, 70, 80, 90 or 95 percent identity in the CDR regions with the CDRregions depicted in the sequences described above.

Since each of these antibodies can bind to IL-13, the V_(H) and V_(L)sequences can be “mixed and matched” to create other anti-IL-13 bindingmolecules of the invention. IL-13 binding of such “mixed and matched”antibodies can be tested using the binding assays described above and inthe Examples (e.g., ELISAs). When V_(H) and V_(L) chains are mixed andmatched, a V_(H) sequence from a particular V_(H)/V_(L) pairing shouldbe replaced with a structurally similar V_(H) sequence. Likewise, aV_(L) sequence from a particular V_(H)/V_(L) pairing should be replacedwith a structurally similar V_(L) sequence. The V_(H) and V_(L)sequences of the antibodies of the present invention are particularlyamenable for mixing and matching, since these antibodies use V_(H) andV_(L) sequences derived from the same germline sequences and thusexhibit structural similarity.

In another aspect, the invention provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of the antibodies, orcombinations thereof. The amino acid sequences of the V_(H) CDR1s of theantibodies are shown in SEQ ID NOs: 6-7. The amino acid sequence of theV_(H) CDR2s of the antibodies is shown by SEQ ID NO: 8. The amino acidsequences of the V_(H) CDR3s of the antibodies are shown in SEQ ID NOs:9-10. The amino acid sequences of the V_(L) CDR1s of the antibodies areshown in SEQ ID NOs: 16-18. The amino acid sequences of the V_(L) CDR2sof the antibodies is shown in SEQ ID NO: 19. The amino acid sequences ofthe V_(L) CDR3s of the antibodies are shown in SEQ ID NOs: 20-22. TheCDR regions are delineated using the Kabat system (Kabat, E. A., et al.,1991 Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242).

Given that each of these antibodies can bind to IL-13 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, although each antibody must contain aV_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3) to create otheranti-IL-13 binding molecules of the invention. IL-13 binding of such“mixed and matched” antibodies can be tested using the binding assaysdescribed above and in the Examples (e.g., ELISAs). When V_(H) CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular V_(H) sequence should be replaced with a structurallysimilar CDR sequence(s). Likewise, when V_(L) CDR sequences are mixedand matched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L)sequence should be replaced with a structurally similar CDR sequence(s).It will be readily apparent to the ordinarily skilled artisan that novelV_(H) and V_(L) sequences can be created by substituting one or moreV_(H) and/or V_(L) CDR region sequences with structurally similarsequences from the CDR sequences shown herein for monoclonal antibodiesof the present invention.

An isolated monoclonal antibody, or antigen binding portion thereof has:a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6-7; a heavy chainvariable region CDR2 comprising an amino acid sequence of SEQ ID NO: 8;a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9-10; a light chainvariable region CDR1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16-18; a light chain variable regionCDR2 comprising an amino acid sequence of SEQ ID NO: 19; and a lightchain variable region CDR3 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 20-22; wherein the antibodyspecifically binds IL-13.

In a certain embodiment, the antibody consists of: a heavy chainvariable region CDR1 comprising SEQ ID NO: 6; a heavy chain variableregion CDR2 comprising SEQ ID NO: 8; a heavy chain variable region CDR3comprising SEQ ID NO: 9; a light chain variable region CDR1 comprisingSEQ ID NO: 16; a light chain variable region CDR2 comprising SEQ ID NO:19; and a light chain variable region CDR3 comprising SEQ ID NO: 20.

In another embodiment, the antibody consists of a heavy chain variableregion CDR1 comprising SEQ ID NO: 7; a heavy chain variable region CDR2comprising SEQ ID NO: 8; a heavy chain variable region CDR3 comprisingSEQ ID NO: 10; a light chain variable region CDR1 comprising SEQ ID NO:17; a light chain variable region CDR2 comprising SEQ ID NO: 19; and alight chain variable region CDR3 comprising SEQ ID NO: 21.

In yet another embodiment, the antibody consists of: a heavy chainvariable region CDR1 comprising SEQ ID NO: 7; a heavy chain variableregion CDR2 comprising SEQ ID NO: 8; a heavy chain variable region CDR3comprising SEQ ID NO: 10; a light chain variable region CDR1 comprisingSEQ ID NO: 18; a light chain variable region CDR2 comprising SEQ ID NO:19; and a light chain variable region CDR3 comprising SEQ ID NO: 22.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or99% identical in amino acid sequence to the amino acid sequence encodedby the germline immunoglobulin gene. Typically, a human antibody derivedfrom a particular human germline sequence will display no more than 10amino acid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the gennlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of the invention has heavy andlight chain variable regions having amino acid sequences that arehomologous to the amino acid sequences of the antibodies describedherein, and wherein the antibodies retain the desired functionalproperties of the anti-IL-13 antibodies of the Invention.

For example, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein: the heavy chainvariable region comprises an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group consistingof SEQ ID NOs: 6-10; the light chain variable region comprises an aminoacid sequence that is at least 80% homologous to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16-22; the antibodyspecifically binds to IL-13, and the antibody exhibits at least one ofthe following functional properties: the antibody inhibits binding IL-13protein to the IL-13 receptor or the antibody inhibits IL-13 receptorbinding preventing or ameliorating an inflammatory or allergiccondition, particularly an inflammatory or obstructive airways disease,or the antibody inhibits IL-13 receptor binding preventing orameliorating asthma or the antibody inhibits IL-13 receptor bindingpreventing or ameliorating COPD.

In various embodiments, the antibody may exhibit one or more, two ormore, or three of the functional properties discussed above. Theantibody can be, for example, a human antibody, a humanized antibody ora chimeric antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homologous to thesequences set forth above. An antibody having V_(H) and V_(L) regionshaving high (i. e., 80% or greater) homology to the V_(H) and V_(L)regions of SEQ ID NOs: 6-10 and 16-22 respectively, can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleicacid molecules encoding SEQ ID NOs: 6-10 and/or 16-22, followed bytesting of the encoded altered antibody for retained function (i. e.,the functions set forth above) using the functional assays describedherein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i. e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat Worldwide Web Site:gcg.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al., 1990 J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997 NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http:llwww.ncbi.nhn.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region consist of CDR1, CDR2, and CDR3 sequences and a lightchain variable region consisting of CDR1, CDR2, and CDR3 sequences,wherein one or more of these CDR sequences have specified amino acidsequences based on the antibodies described herein or conservativemodifications thereof, and wherein the antibodies retain the desiredfunctional properties of the anti-IL-13 antibodies of the invention.Accordingly, the invention provides an isolated monoclonal antibody, orantigen binding portion thereof, consisting of a heavy chain variableregion consisting of CDR1, CDR2, and CDR3 sequences and a light chainvariable region consisting of CDR1, CDR2, and CDR3 sequences, wherein:the heavy chain variable regions of CDR1 is sequences consisting ofamino acid sequences selected from the group consisting of amino acidsequences of SEQ ID NOs: 6-7, and conservative modifications thereof;the heavy chain variable region of CDR2 is a sequence consisting of anamino acid sequence of SEQ ID NO: 8, and conservative modificationsthereof; the heavy chain variable region of CDR3 is sequences consistingof amino acid sequences selected from the group consisting of amino acidsequences of SEQ ID NOs: 9-10, and conservative modifications thereof;the light chain variable regions of CDR1 is sequences consisting ofamino acid sequences selected from the group consisting of amino acidsequences of SEQ ID NOs: 16-18, and conservative modifications thereof;the light chain variable regions of CDR2 is a sequence consisting of anamino acid sequence of SEQ ID NO: 19, and conservative modificationsthereof; the light chain variable regions of CDR3 is sequencesconsisting of amino acid sequences selected from the group consisting ofamino acid sequences of SEQ ID NOs: 20-22, and conservativemodifications thereof; the antibody specifically binds to IL-13; and theantibody inhibits IL-13 receptor binding preventing inflammatorymediator release.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, scrim, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-IL-13 Antibodies of theInvention

In another embodiment, the invention provides antibodies that bind tothe same epitope as do the various anti-IL-13 antibodies of theinvention provided herein. Such additional antibodies can be identifiedbased on their ability to cross-compete (e.g., to competitively inhibitthe binding of, in a statistically significant manner) with otherantibodies of the invention in standard IL-13 binding assays. Theability of a test antibody to inhibit the binding of antibodies of thepresent invention to human IL-13 demonstrates that the test antibody cancompete with that antibody for binding to human IL-13; such an antibodymay, according to non-limiting theory, bind to the same or a related(e.g., a structurally similar or spatially proximal) epitope on humanIL-13 as the antibody with which it competes. In a certain embodiment,the antibody that binds to the same epitope on human IL-13 as theantibodies of the present invention is a human monoclonal antibody. Suchhuman monoclonal antibodies can be prepared and isolated as described inthe Examples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i. e., V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or antigen binding portion thereof, comprising aheavy chain variable region comprising CDR1 sequences having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6-7;CDR2 sequences having an amino acid sequence of SEQ ID NO: 8; CDR3sequences having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 9-10, respectively; and a light chain variableregion having CDR1 sequences having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 16-18; CDR2 sequences having anamino acid sequence of SEQ ID NO: 19; and CDR3 sequences consisting ofan amino acid sequence selected from the group consisting of SEQ ID NOs:20-22, respectively. Thus, such antibodies contain the V_(H) and V_(L)CDR sequences of monoclonal antibodies, yet may contain differentframework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at Worldwide Web Site:mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al., 1991Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798; and Cox, J. P.L. et al., 1994 Eur. J Immunol. 24:827-836; the contents of each ofwhich are expressly incorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2and 3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolatedanti-IL-13 monoclonal antibodies, or antigen binding portions thereof,consisting of a heavy chain variable region having: a V_(H) CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 6-7 or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 6-7; a V_(H) CDR2 region having an amino acid sequence of SEQ IDNO: 8, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNO: 8; a V_(H) CDR3 region having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 9-10, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 9-10; a V_(L) CDR1 region havingan amino acid sequence selected from the group consisting of SEQ ID NOs:16-18, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 16-18; a V_(L) CDR2 region having an amino acid sequence of SEQ IDNO: 19, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNO: 19; and a V_(L) CDR3 region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 20-22, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 20-22.

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to bo encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell—epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Can et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glyeosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et at.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodrneret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen’. Suchcarbohydrate modifications can be accomplished by; for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half-life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.The pegylation can be carried out by an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

As discussed above, the anti-IL-13 antibodies having V_(H) and V_(L)sequences shown herein can be used to create new anti-IL-13 antibodiesby modifying the V_(H) and/or V_(L) sequences, or the constant region(s)attached thereto. Thus, in another aspect of the invention, thestructural features of an anti-IL-13 antibody of the invention are usedto create structurally related anti-IL-13 antibodies that retain atleast one functional property of the antibodies of the invention, suchas binding to human IL-13 and also inhibiting one or more functionalproperties of IL-13 (e.g., receptor binding, inhibition of mediatorrelease).

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-IL-13 antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-IL-13 antibody consisting of: a heavy chain variableregion antibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 6-7, a CDR2 sequence of SEQ ID NO: 8 and/or aCDR3 sequence selected from the group consisting of SEQ ID NOs: 9-10;and a light chain variable region antibody sequence having a CDR1sequence selected from the group consisting of SEQ ID NOs: 16-18, a CDR2sequence of SEQ ID NO: 19 and/or a CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 20-22; altering at least one amino acidresidue within the heavy chain variable region antibody sequence and/orthe light chain variable region antibody sequence to create at least onealtered antibody sequence; and expressing the altered antibody sequenceas a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the anti-IL-13 antibodies described herein,which functional properties include, but are not limited to,specifically binding to human IL-13; and the antibody exhibits at leastone of the following functional properties: the antibody inhibitsbinding of IL-13 protein to the IL-13 receptor, or the antibody inhibitsIL-13 receptor binding preventing or ameliorating an inflammatory,fibrotic or allergic condition, particularly an inflammatory orobstructive airways disease, or the antibody inhibits IL-13 receptorbinding thereby preventing or ameliorating asthma.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-IL-13 antibody coding sequence and the resultingmodified anti-IL-13 antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. The nucleic acids may be presentin whole cells, in a cell lysate, or may be nucleic acids in a partiallypurified or substantially pure form. A nucleic acid is “isolated” or“rendered substantially pure” when purified away from other cellularcomponents or other contaminants, e.g., other cellular nucleic acids orproteins, by standard techniques, including alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis and otherswell known in the art. See, F. Ausubel, et al., ed. 1987 CurrentProtocols in Molecular Biology, Greene Publishing and WileyInterscience, New York. A nucleic acid of the invention can be, forexample, DNA or RNA and may or may not contain intronic sequences. In anembodiment, the nucleic acid is a cDNA molecule. The nucleic acid may bepresent in a vector such as a phage display vector, or in a recombinantplasmid vector.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromvarious phage clones that are members of the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to an scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA molecule, or to a fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked”, as used in this context, isintended to mean that the two DNA fragments are joined in a functionalmanner, for example, such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame, or such that the protein is expressedunder control of a desired promoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., el al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragmentheavy chain gene, the V_(H)-encoding DNA can be operatively linked toanother DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991 Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., 1988 Science 242:423-426; Huston et at., 1988 Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature348:552-554).

Production of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstIL-13 can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N.Y. Acad. Sci. 764:536-546). The preparation anduse of HuMAb mice, and the genomic modifications carried by such mice,is further described in Taylor, L. et al., 1992 Nucleic Acids Research20:6287-6295; Chen, J. et at., 1993 International Immunology 5: 647-656;Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724; Choi etal., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBO J. 12:821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor, L. etal., 1994 International Immunology 579-591; and Fishwild, D. et al.,1996 Nature Biotechnology 14: 845-851, the contents of all of which arehereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IL-13 antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-IL-13 antibodies of the invention. For example, mice carrying botha human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raise anti-IL-13antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Generation of Human Monoclonal Antibodies Against IL-13

Purified recombinant human (hr) IL-13 conjugated to Pan DR T helperEpitopes (PADRE), is used as the antigen. Fully human monoclonalantibodies to IL-13 are prepared using HCo7 strains of HuMab transgenicmice which express human antibody genes. In this mouse strain, theendogenous mouse kappa light chain gene can be homozygously disrupted asdescribed in Chen et al., 1993 EMBO J. 12:811-820 and the endogenousmouse heavy chain gene can be homozygously disrupted as described inExample 1 of PCT Publication WO 01109187. This mouse strain carries ahuman kappa light chain transgene, KCo5, as described in Fishwild etal., 1996 Nature Biotechnology 14:845-851 and the HCo7 human heavy chaintransgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and5,545,807.

To generate fully human monoclonal antibodies to IL-13 of the invention,HuMab mice are immunized with a mixture of purified recombinant IL-13derived from HEK-EBNA/PADRE conjugate (42 ug/mouse) and Quil A (15ug/mouse, Accurate Chemical). General immunization schemes for HuMabmice are described in Lonberg, N. et al., 1994 Nature 368(6474):856-859; Fishwild, D. et al., 1996 Nature Biotechnology 14:845-851 andPCT Publication WO 98/24884. Transgenic mice are immunized eitherintravenously (IV), or subcutaneously (SC) between day 1-71. Mice areboosted intravenously with antigen (without adjuvant) 2 days beforesacrifice and removal of the spleen. RNA was isolated from spleens usingthe Nucleospin RNA II isolation kit (BD Biosciences/Clontech). The RNAwas used to generate a phage display library of randomly assorted H andL chain variable domains in a Fab phage display vector as described inU.S. Pat. No. 6,794,132. The phage display library was subjected to fiverounds of selection using biotinylated hrIL-13 in a solution-phaseequilibrium binding protocol as described in the patent. The first fourrounds of selection employed hrIL-13 at 10⁻⁸ M and the last round ofselection employed hrIL-13 at 10⁻⁹ M. The final signal to noise ratiodetermined by counting pfu's recovered in the presence of antigendivided by pfu's recovered in the absence of antigen was 37 for thislibrary, indicating that greater than 90% of the phage selected wereexpressing antibodies that bound hrIL-13. The phage display library wasthen subcloned into a plasmid vector for the expression of soluble Fabas described in U.S. Pat. No. 6,794,132.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. in this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the CH segment(s) within the vector andthe V_(L) segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, inparticular mammalian host cells, is discussed because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today6:12-13).

Mammalian host cells for expressing the recombinant antibodies of theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp, 1982 Mol. Biol. 159:601-621, NSO myelomacells, COS cells and SP2 cells. When recombinant expression vectorsencoding antibody genes are introduced into mammalian host cells, theantibodies are produced by culturing the host cells for a period of timesufficient to allow for expression of the antibody in the host cells orsecretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Immunoconjugates

In another aspect, the present invention features an anti-IL-13antibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a cytotoxin, a drug (e.g., an immunosuppressant) or aradiotoxin. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates that include one or more cytotoxinsare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), ablating agents (e.g., mechlorethamine, thioepachloraxnbucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody of the invention include duocarmycins, calicheamicins,maytansines and auristatins, and derivatives thereof. An example of acalicheamicin antibody conjugate is commercially available (Mylotarg™;Wyeth-Ayerst).

Cytotoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.,2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al., 2003 CancerImmunol. Immunother. 52:328-337; Payne, G., 2003 Cancer Cell 3:207-212;Allen, T. M., 2002 Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman,R. J., 2002 Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P. D. andSpringer, C. J., 2001 Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰, and lutetium¹⁷⁷. Method for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (DEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et at., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Inmunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-IL-13 antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may in fact be derivatized or linked to more than one otherfunctional molecule to generate multi-specific molecules that bind tomore than two different binding sites and/or target molecules; suchmulti-specific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for IL-13 and a secondbinding specificity for a second target epitope. For example, the secondtarget epitope is an Fc receptor, e.g., human FcγRI (CD64) or a humanFcα receptor (CD89). Therefore, the invention includes bispecificmolecules capable of binding both to FcγR, FcαR or FcεR expressingeffector cells (e.g., monocytes, macrophages or polymorphonuclear cells(PMNs), and to target cells expressing IL-13. These bispecific moleculestarget IL-13 expressing cells to effector cell and trigger Fcreceptor-mediated effector cell activities, such as phagocytosis of anIL-13 expressing cells, antibody dependent cell-mediated cytotoxicity(ADCC), cytokine release, or generation of superoxide anion.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-IL-13 binding specificity. For example, the third bindingspecificity could be an anti-enhancement factor (EF) portion, e.g., amolecule which binds to a surface protein involved in cytotoxic activityand thereby increases the immune response against the target cell. The“anti-enhancement factor portion” could be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor, and thereby results in an enhancement of theeffect of the binding determinants for the Fc receptor or target cellantigen.

The “anti-enhancement factor portion” can bind an Fc receptor or atarget cell antigen. Alternatively, the anti-enhancement factor portioncould bind to an entity that is different from the entity to which thefirst and second binding specificities bind. For example, theanti-enhancement factor portion can bind a cytotoxic T-cell (e.g. byCD2, CD3, CD8, CD28, CD4, CD44, ICAM-1 or other immune cell that resultsin an increased immune response against the target cell).

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof; including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fγ receptor classes: FcγRI (CD64),FcγRII (CD32), and FcγRIII (CD 16). In another embodiment, the Fcγreceptor is a human high affinity FcγRI. The human FcγRI is a 72 kDamolecule, which shows high affinity for monomeric IgG (10⁸-10⁹ M⁻¹).

The production and characterization of certain anti-Fcγ monoclonalantibodies are described by Fanger et at. in PCT Publication WO 88/00052and in U.S. Pat. No. 4,954,617, the teachings of which are fullyincorporated by reference herein. These antibodies bind to an epitope ofFcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγbinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al., 1995 J. Immunol 155(10): 4996-5002 and PCT Publication WO 94/10332. The 1122 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other embodiments, the binding specificity for an Fc receptoris provided by an antibody that binds to a human IgA receptor, e.g., anFc-alpha receptor (FcαRI (CD89), the binding of which does not have tobe blocked by human immunoglobulin A (IgA). The term “IgA receptor” isintended to include the gene product of one a gene (FcαRI) located onchromosome 19. This gene is known to encode several alternativelyspliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) isconstitutively expressed on monocytes/macrophages, eosinophilic andneutrophilic granulocytes, but not on non-effector cell populations.FcαRI has medium affinity (5×10⁷ M⁻¹) for both IgA1 and IgA2, which isincreased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al., 1996 Critical Reviews in Immunology 116:423-440). FourFcαRI-specific monoclonal antibodies, identified as A3, A59, A62 andA77, which bind FcαRI outside the IgA ligand binding domain, have beendescribed (Monteiro, R. C. et al., 1992 J. Immunol. 148:1764).

FcαRI and FcγRI are trigger receptors for use in the bispecificmolecules of the invention because they are expressed primarily onimmune effector cells, e.g., monocytes, PMNs, macrophages and dendriticcells; expressed at high levels (e.g., 5,000-100,000 per cell);mediators of cytotoxic activities (e.g., ADCC, phagocytosis); mediateenhanced antigen presentation of antigens, including self-antigens,targeted to them.

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-IL-13 binding specificities, using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-l-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunel. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactively 4labeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub; B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-IL-13 antibody of the presentinvention combined with at least one other anti-inflammatory agent.Examples of therapeutic agents that can be used in combination therapyare described in greater detail below in the section on uses of theantibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al., 1977 J. Pharm. Sci. 66: 1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, one can include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption for example, monostearatesalts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, from about 0.1percent to about 70 percent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Dosage regimens for an anti-IL-13 antibody ofthe invention may include 1 mg/kg body weight or 3 mg/kg body weight byintravenous or subcutaneous administration, with the antibody beinggiven using one of the following dosing schedules: e.g. every four weeksfor six dosages, then every three months; every three weeks; 3 mg/kgbody weight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-IL-13 antibody of theinvention can results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.

A composition of the present invention can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the inventioninclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrastemal injection andinfusion.

Alternatively, an antibody of the invention can be administered by anonparenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices shown in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.Examples of well known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which shows an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which shows a therapeutic device for administeringmedicants through the skin; U.S. Pat. No. 4,447,233, which shows amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which shows a variable flow implantableinfusion apparatus for continuous drug delivery; U.S. Pat. No.4,439,196, which shows an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which shows anosmotic drug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade,1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M.Owais et al., 1995 Antimicrob. Agents Chernother. 39: 180); surfactantprotein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120(Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen, 1994 FEBS Lett. 346:123; J. J. Killion; I. J. Fidler,1994 Immunomethods 4:273.

Uses and Methods of the Invention

The antibodies (and immunoconjugates and bispecific molecules) of thepresent invention have in vitro and in vivo diagnostic and therapeuticutilities. For example, these molecules can be administered to cells inculture, e.g. in vitro or in vivo, or in a subject, e.g., in vivo, totreat, prevent or diagnose a variety of disorders. The term “subject” asused herein in intended to include human and non-human animals.Non-human animals includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, cows,horses, chickens, amphibians, and reptiles. The methods are particularlysuitable for treating human patients having a disorder associated withaberrant IL-13 expression. When antibodies to IL-13 are administeredtogether with another agent, the two can be administered in either orderor simultaneously.

In one embodiment, the antibodies (and immunoconjugates and bispecificmolecules) of the invention can be used to detect levels of IL-13, orlevels of cells that contain IL-13. This can be achieved, for example,by contacting a sample (such as an in vitro sample) and a control samplewith the anti-IL-13 antibody under conditions that allow for theformation of a complex between the antibody and IL-13. Any complexesformed between the antibody and IL-13 are detected and compared in thesample and the control. For example, standard detection methods, wellknown in the art, such as ELISA and flow cytometic assays, can beperformed using the compositions of the invention.

Accordingly, in one aspect, the invention further provides methods fordetecting the presence of IL-13 (e.g., human IL-13 antigen) in a sample,or measuring the amount of IL-13, comprising contacting the sample, anda control sample, with an antibody of the invention, or an antigenbinding portion thereof, which specifically binds to IL-13, underconditions that allow for formation of a complex between the antibody orportion thereof and IL-13. The formation of a complex is then detected,wherein a difference in complex formation between the sample compared tothe control sample is indicative of the presence of IL-13 in the sample.

Also within the scope of the invention are kits consisting of thecompositions (e.g., antibodies, human antibodies, immunoconjugates andbispecific molecules) of the invention and instructions for use. The kitcan further contain a least one additional reagent, or one or moreadditional antibodies of the invention (e.g., an antibody having acomplementary activity which binds to an epitope on the target antigendistinct from the first antibody). Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

The invention having been fully described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references, including issued patents and publishedpatent applications, cited throughout this application are herebyincorporated by reference.

EXAMPLES Example 1 Generation of Human IL-13-Specific Antibodies fromImmunized Splenic Libraries

The RNA from spleen was used to generate a phage display library ofrandomly assorted H and L chain variable domains in a Fab phage displayvector as described in U.S. Pat. No. 6,794,132. The phage displaylibrary was subjected to five rounds of selection using biotinylatedhrIL-13 in a solution-phase equilibrium binding protocol as described inthe patent. The first four rounds of selection employed hrIL-13 at 10⁻⁸M and the last round of selection employed hrIL-13 at 10⁻⁹ M. The finalsignal to noise ratio determined by counting pfu's recovered in thepresence of antigen divided by pfu's recovered in the absence of antigenwas 37 for this library, indicating that greater than 90% of the phageselected were expressing antibodies that bound hrIL-13. The phagedisplay library was then subcloned into a plasmid vector for theexpression of soluble Fab as described in U.S. Pat. No. 6,794,132. Thesubcloned is library comprises plasmid vectors in E. coli, each plasmidencoding a monoclonal Fab fragment. The subclone library was plated outand colonies representing individual clones were picked to inoculate96-well plates. After overnight growth, the plate cultures were used toarchive frozen cell banks for the clones in 96-well plates and also toseed replica 96-well plates which were induced to express monoclonalantibodies. The next day, these 96-well plate cultures were subjected todetergent extraction and purification to recover microgram quantities ofantibodies. The purified antibodies were treated to remove endotoxin andsterile filtered terminally. ELISA assays were conducted usingbiotinylated rhIL-13 coated on avidin plates to identify which wellscontained functional positives. Sandwich assays targeting the constantregion of the antibodies were used to determine antibody concentrationsin different wells. The 96-well plates containing the antibodies and theassay data were assessed for biological activity. The clones of interestin the 96-well frozen cell banks were then sequenced to identify theones expressing unique antibodies. Subsequently, the frozen cell banksof these unique clones were used to seed small scale shake flaskcultures and grown overnight. Large scale flasks were seeded using theovernight cultures and then induced to express antibodies. The next day,the flask cultures were mechanically homogenized and purified to yieldmilligram quantities of antibodies. The purified Fabs were processed forendotoxin removal and subjected to terminal sterile filtration. Thefunctional activity of these antibodies was demonstrated by ELISA usingbiotinylated rhIL-13 coated on avidin plates. The antibodyconcentrations were determined by absorbance measurement at 280 nm. Thepurified Fabs were assessed for in vitro binding and activity in a cellbased assay.

Example 2 Quantitative Analysis of Binding Affinities: Determination ofAnti-Human IL-13 Fab Candidates

Surface plasmon resonance measurements quantifying the interaction ofanti-IL-13 Fabs with several hrIL-13 are performed using the opticalbiosensor, BIAcore 2000. Specific binding of IL-13 to a respective IL-13Fab immobilized on a BlAcore chip can be measured by followingaccumulation of the ligand on the receptor. The microscopic association(k_(on)) and dissociation rates (k_(off)) can be obtained directly fromthe mass accumulation rates on the chip and are expressed in responseunits (RUs). Anti-IL-13 Fab is immobilized on the chip surface via asecondary anti-human Lκ antibody (Jackson Immunochemicals). This captureantibody was covalently bound using the ‘Amine coupling kit’ (BIAcore,Cat. No. BR-1000-50) as recommended in the manufacturers' protocols. 250μl of varying concentrations of hrIL-13 was injected at flow rate of 20μl/min and the kinetic trace was recorded. The chip surface wasregenerated by two acid wash steps using 100 mM HCl and injecting 10 μlwith a flow rate of 20 μl/min. This treatment leads to dissociation ofthe Fab IL-13 complex due to reversible acid denaturation. Nosignificant loss of binding activity was observed when the antibody wasreinjected for a subsequent run. The kinetic traces were evaluated withthe BIAcore software applying the 1:1 Langmuir association model Thesummarized affinity data on human IL-13 is shown in Table 1 herein.

TABLE I Fab KD [pM] human IL-13 01471/G6 100 ± 2 03161/H2 197 ± 1201951/G12 480 ± 68 01771/E10 343 ± 54

Example 3 Conversion into the IgG Format

Antibody DNA sequencing templates were purified from 3 ml cultures usingQIAprep minipreps (Qiagen inc.). Templates were sequenced using anApplied Biosystems 3100 Avant Genetic Analyzer according tomanufacturer's specifications. The heavy and kappa chain variableregions of selected clones were separately amplified from the sequencingtemplates by PCR, purified by agarose gel electrophoresis, and excisedand purified from the gel. Plasmids encoding the V_(H) and V_(L) werecloned into expression cassettes for human kappa light and human IgG₁heavy chains. The Sp2/0 parental cell line was transfected with twovectors, one for the fight and one for heavy chain vectors. Transfectedcells were selected and amplified using G418 and methotrexaterespectively resulting in the emergence of resistant, amplified cellpools producing the antibodies with titers ranging from 5 mg/L to 30mg/L. Dilution cloning was then employed, resulting in the isolation of127 viable clones from six 96-well plates. Emerging cell lines were thentested for productivity in a fed-batch shaker format. Stability testingto confirm the stable integration of the expression constructs androbust expression of product were also performed over a 90-day period.Northern blots exhibited single, full length RNAs with equal bandintensities, indicating similar expression levels for both the heavychain and light chain.

Example 4 In Vitro Characterization of Anti-IL-13 Full Antibodies in aCell Based Assay

IL-13 is a potent inducer of eotaxin release from human lungfibroblasts. The ability of the antibodies to neutralize the bioactivityof IL-13 was assessed in an IL-13-induced eotaxin release assay usinghuman lung fibroblasts. Briefly, cells (2×10⁴ cells per well in a volumeof 100 μl) were plated out in each well of a 96 well tissue cultureplate. The cells were stimulated with a concentration of IL-13conferring 80% of the maximal eotaxin release, which was pre-determinedfor each batch of cells using a standard curve of 0-100 ng/ml IL-13.Varying concentrations of the antibodies were co-applied to the cells.The cells were allowed to incubate for 24 h at 37° C., 5% CO₂ and theculture media was harvested and stored at −20° C. until required.Eotaxin levels within the media were measured by specific ELISA (R&Dsystems) where the sensitivity of assay was between 15-1000 pg/ml.

The anti-IL-13 Fabs were thereby analyzed with respect to EC₅₀ asdescribed above and shown in Table 2.

TABLE 2 Antibody EC₅₀ [nM] human IL-13 01471/G6 1.23 ± 0.4 03161/H2 0.95± 0.2 01951/G12 0.33 ± 0.1 01771/E10 1.71 ± 0.5

Example 5 Sequence Analysis of the Anti-IL-13 Antibodies

The nucleotide sequences of the heavy and light chain variable regions(V_(H) and V_(L)) of all antibodies were determined. Amino acidsequences of the complementarity determining regions (CDRs) are listedin Table 3 and 4 herein. The CDRs according Kabat definition (E. Kabatet al, 1991, Sequences of Proteins of Immunological Interest, 5thedition, Public Health Service, National Institute of Health, Bethesda,Md., are listed in Table 3a and 4a.

TABLE 3 SEQ SEQ SEQ ID ID ID No. No. No. Antibody HCDR1 HCDR1 HCDR2HCDR2 HCDR3 HCDR3 01471/G6 GFTFSNYG 1 IWYDGSN 3 VKGSGDIP 4 03161/H2GFTFSNYG 1 IWYDGSN 3 VKGSGDIP 4 01951/G12 GFTFSSYG 2 IWYDGSN 3 ARLWFGDLD5 01771/E10 GFTFSSYG 2 IWYDGSN 3 ARLWFGDLD 5

TABLE 3a SEQ ID No. SEQ ID No. SEQ ID No. Antibody HCDRI HCDR1 HCDR2HCDR2 HCDR3 HCDR3 01471/G6 NYGMH 8 IIWYDGSNKYYADSVKG 8 GSGDIPFDY 903161/H2 NYGMH 6 IIWYDGSNKYYADSVKG 8 GSGDIPFDY 9 01951/G12 SYGMH 7IIWYDGSNKYYADSVKG 8 LWFGDLDAFDI 10 01771/E10 SYGMH 7 IIWYDGSNKYYADSVKG 8LWFGDLDAFDI 10

TABLE 4 SEQ SEQ SEQ ID ID ID  No. No. No. Antibody LCDR1 LCDR1 LCDR2LCDR2 LCDR3 LCDR3 01471/G6 QSVSSY 11 DA 12 HQRSHWPPI 13 03161/H2 QSVSSY11 DA 12 HQRSHWPPI 13 01951/G12 QSVSSY 11 DA 12 QQRSSWPPV 14 01771/E10QSVSSY 11 DA 12 HQRSSWPPI 15

TABLE 4a SEQ ID No. SEQ ID No. SEQ ID No. Antibody LCDR1 LCDR1 LCDR2LCDR2 LCDR3 LCDR3 01471/G6 RASQSVSSYLA 16 DASNRAT 19 HQRSHWPPIFT 2003161/H2  RASQSVSSYLA 16 DASNRAT 19 HQRSHWPPIFT 20 01951/G12 RAGQSVSSYLV17 DASNRAT 19 QORSSWPPVYT 21 01771/E10 RASQSVSSYLA 18 DASNRAT 19HQRSSWPPIFT 22

The sequences of the antibodies of the previous tables, includingframework regions, are shown below. The full IgG1 antibody light andheavy chain constant regions are also shown below, incorporating, as anexample, the variable regions of antibody 01951/G12 (emboldened).

01471/G6 Antibody Sequence

(i) HC Variable Region

The HC variable amino acid sequence for 01471/G6 is shown in SEQ ID NO:23 and is encoded by the nucleotide sequence shown in SEQ ID NO: 24

E  V  Q  L  V  E  S  G  G  G  V  V  Q  P  G  R  S  L  R  L(SEQ ID NO: 23)gaagtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60(SEQ ID NO: 24)S  C  A  A  S  G  F  T  F  S  N  Y  G  M  H  W  V  R  Q  Atcctgtgcagcgtctggattcaccttcagtaactatggcatgcactgggtccgccaggct 120P  G  K  G  L  E  W  V  A  I  I  W  Y  D  G  S  N  K  Y  Yccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180A  D  S  V  K  G  R  F  T  I  S  R  D  N  S  K  N  T  L  Ygcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat 240L  Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  V  K  G  Sctgcaaatgaacagtctgagagccgaggacacggctgtgtattactgtgtgaaaggatct 300G  D  I  P  F  D  Y  W  G  Q  G  T  L  V  Tggggatattccctttgactactggggccagggaaccctggtcacc 345(ii) LC Variable Region

The LC variable amino acid sequence for 01471/G6 is shown in SEQ ID NO:25 and is encoded by the nucleotide sequence shown in SEQ ID NO: 26

E  I  V  L  T  Q  S  P  A  T  L  S  S  S  P  G  E  R  A  T(SEQ ID NO: 25)gaaattgtgttgacgcagtctccagccaccctgtcttcgtctccaggggaaagagccacc 60(SEQ ID NO: 26)L  S  C  R  A  S  Q  S  V  S  S  Y  L  A  W  Y  Q  Q  K  Pctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct 120G  Q  A  P  R  L  L  I  Y  D  A  S  N  R  A  T  G  I  P  Aggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc 180R  F  S  G  S  G  S  G  T  D  F  T  L  T  I  S  S  L  E  Paggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240E  D  F  A  V  Y  Y  C  H  Q  R  S  H  W  P  P  I  F  T  Fgaagattttgcagtctattactgtcatcagcgtagccactggcctcccatattcactttc 300G  P  G  T ggccctgggacc 31203161/H2 Antibody(i) HC Variable Region

The HC variable amino acid sequence for 03161/H2 is shown in SEQ ID NO:27 and is encoded by the nucleotide sequence shown in SEQ ID NO: SEQ IDNo. 28

E  V  Q  L  V  E  S  G  G  G  V  V  Q  P  G  R  S  L  R  L(SEQ ID NO: 27)gaagtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactc  60(SEQ ID NO :28)S  C  A  A  S  G  F  T  F  S  N  Y  G  M  H  W  V  R  Q  Atcctgtgcagcgtctggattcaccttcagtaactatggcatgcactgggtccgccaggct 120P  G  K  G  L  E  W  V  A  I  I  W  Y  D  G  S  N  K  Y  Yccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180A  D  S  V  K  G  R  F  T  I  S  R  D  N  S  K  N  T  L  Ygcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat 240L  Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  V  K  G  Sctgcaaatgaacagtctgagagccgaggacacggctgtgtattactgtgtgaaaggatct 300G  D  I  P  F  D  Y  W  G  Q  G  T  L  V  Tggggatattccctttgactactggggccagggaaccctggtcacc 345>(ii) LC Variable Region

The LC variable amino acid sequence for 03161/H2 is shown in SEQ ID NO:29 and is encoded by the nucleotide sequence shown in SEQ ID NO: 30

E  I  V  L  T  Q  S  P  A  T  L  S  S  S  P  G  E  R  A  T(SEQ ID NO: 29)gaaattgtgttgacgcagtccccagccaccctgtcttcgtctccaggggaaagagccacc 60(SEQ ID NO: 30)L  S  C  R  A  S  Q  S  V  S  S  Y  L  A  W  Y  Q  Q  K  Pctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct 120G  Q  A  P  R  L  L  I  Y  D  A  S  N  R  A  T  G  T  P  Aggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcaccccagcc 180R  F  S  G  S  G  S  G  T  D  F  T  L  T  I  S  S  L  E  Paggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240E  D  F  A  V  Y  Y  C  H  Q  R  S  H  W  P  P  I  F  T  Fgaagattttgcagtctattactgtcatcagcgtagccactggcctcccatattcactttc 300G  P  G  T ggccctgggacc 31201951/G12 Antibody Sequence(i) HC Variable Region

The HC variable amino acid sequence for 01951/G12 is shown in SEQ ID NO:31 and is encoded by the nucleotide sequence shown in SEQ ID NO: 32

E  V  Q  L  V  E  S  G  G  G  V  V  Q  P  G  R  S  L  R  L(SEQ ID NO: 31)gaagtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60(SEQ ID NO: 32)S  C  A  A  S  G  F  T  F  S  S  Y  G  M  H  W  V  R  Q  Atcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggct 120P  G  K  G  L  E  W  V  A  I  I  W  Y  D  G  S  N  K  Y  Yccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180A  D  S  V  K  G  R  F  T  I  S  R  D  N  S  K  N  T  L  Ygcggactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat 240L  Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  A  R  L  Wctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggctatgg 300F  G  D  L  D  A  F  D  I  W  G  Q  G  T  M  V  Tttcggggacttagatgcttttgatatctggggccaagggacaatggtcacc 351(ii) LC Variable Region

The LC variable amino acid sequence for 01951/G12 is shown in SEQ ID NO:33 and is encoded by the nucleotide sequence shown in SEQ ID NO: 34

E  I  V  L  T  Q  S  P  A  T  L  S  L  S  P  G  E  R  A  I(SEQ ID NO: 33)gaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccatc 60(SEQ ID NO: 34)L  S  G  R  A  G  Q  S  V  S  S  Y  L  V  W  Y  Q  Q  K  Pctctcctgcagggccggtcagagtgttagcagttacttagtctggtaccaacagaaacct 120G  Q  A  P  R  L  L  I  Y  D  A  S  N  R  A  T  G  I  P  Aggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc 180R  F  S  G  S  G  S  G  T  D  F  T  L  T  I  S  S  L  E  Paggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240E  D  F  A  V  Y  Y  C  Q  Q  R  S  S  W  P  P  V  Y  T  Fgaagattttgcagtttattactgtcagcagcgcagcagctggcctccggtgtacactttt 300G  Q  G  T ggccaggggacc 31201771/E10 Antibody Sequence(i) HC Variable Region

The HC variable amino acid sequence for 01771/E10 is shown in SEQ ID NO:35 and is encoded by the nucleotide sequence shown in SEQ ID NO: 36

Q  V  Q  L  V  Q  S  G  G  G  V  V  Q  P  G  R  S  L  R  L(SEQ ID NO: 35)caggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60(SEQ ID NO: 36)S  C  A  A  S  G  F  T  F  S  S  Y  G  M  H  W  V  R  Q  Atcctgtgcggcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggct 120P  G  K  G  L  E  W  V  A  I  I  W  Y  D  G  S  N  K  Y  Yccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180A  D  S  V  K  G  R  F  T  I  S  R  D  N  S  K  N  T  L  Ygcggactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctatat 240L  Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  A  R  L  Wctacaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggctatgg 300F  G  D  L  D  A  F  D  I  W  G  Q  G  T  M  V  Tttcggggacttagatgcttttgatatctggggccaagggacaatggtcacc 351(ii) LC Variable Region

The LC variable amino acid sequence for 01771/E10 is shown in SEQ ID NO:37 and is encoded by the nucleotide sequence shown in SEQ ID NO: 38

E  I  V  L  T  Q  S  P  A  T  L  S  L  S  P  G  E  R  A  T(SEQ ID NO: 37)gaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccacc 60(SEQ ID NO: 38)L  S  C  R  A  S  Q  S  V  S  S  Y  L  A  W  Y  Q  Q  K  Pctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct 120G  Q  A  P  R  L  L  I  Y  D  A  S  N  R  A  T  G  I  P  Aggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc 180R  F  S  G  S  G  S  G  T  D  F  T  L  T  I  S  S  L  E  Paggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240E  D  F  A  V  Y  Y  C  H  Q  R  S  S  W  P  P  I  F  T  Fgaagattttgcggtttattactgtcatcagcgtagcagctggcccccgatattcactttc 300G  P  G  T ggccctgggacc 312Full Antibody IgG1 Light Chain Sequence Incorporating the VariableRegion of Antibody 01951/G12 (Emboldened)

The LC amino acid sequence is shown in SEQ ID NO: 39 and is encoded bythe nucleotide sequence of SEQ ID NO: 40

M  S  V  L   T  Q  V   L  A  L   L  L  L  W   L  T  G (SEQ ID NO: 39)  1 ATGAGTGTGC TCACTCAGGT CCTGGCGTTG CTGCTGCTGT GGCTTACAGG(SEQ ID NO: 40)  T  R  C   E  I  V  L   T  Q  S   P  A  T   L  S  L  S 51 TACGCGTTGT GAAATTGTGT TGACGCAGTC TCCAGCCACC CTGTCTTTGT  P  G  E   R  A  I   L  S  C  R   A  G  Q   S  V  S 101CTCCAGGGGA AAGAGCCATC CTCTCCTGCA GGGCCGGTCA GAGTGTTAGCS  Y  L  V   W  Y  Q   Q  K  P   G  Q  A  P   R  L  L 151AGTTACTTAG TCTGGTACCA ACAGAAACCT GGCCAGGCTC CCAGGCTCCT I  Y  D   A  S  N  R   A  T  G   I  P  A   R  F  S  G 201CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG  S  G  S   G  T  D   F  T  L  T   I  S  S   L  E  P 251GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCTE  D  F  A   V  Y  Y   C  Q  Q   R  S  S  W   P  P  V 301GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGCAGCAGCT GGCCTCCGGT Y  T  F   G  Q  G  T   K  L  E   I  K  R   T  V  A  A 351GTACACTTTT GGCCAGGGGA CCAAGCTTGA AATCAAACGA ACTGTGGCTG  P  S  V   F  I  F   P  P  S  D   E  Q  L   K  S  G 401CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGAT  A  S  V   V  C  L   L  N  N   F  Y  P  R   E  A  K 451ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA V  Q  W   K  V  D  N   A  L  Q   S  G  N   S  Q  E  S 501AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA  V  T  E   Q  D  S   K  D  S  T   Y  S  L   S  S  T 551GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACCL  T  L  S   K  A  D   Y  E  K   H  K  V  Y   A  C  E 601CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAAGTCT ACGCCTGCGA V  T  H   Q  G  L  S   S  P  V   T  K  S   F  N  R  G 651AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG   E  C  * 701GAGAGTGTTA GFull Antibody IgG1 Heavy Chain Sequence Incorporating the VariableRegion of Antibody 01951/G12 (Emboldened)

The HC amino acid sequence is shown in SEQ ID NO: 41 and is encoded bythe nucleotide sequence of SEQ ID NO: 42

M  A  W  V   W  T  L   P  F  L   M  A  A  A   Q  S  V (SEQ ID NO: 41)   1 ATGGCTTGGG TGTGGACCTT GCCATTCCTG ATGGCAGCTG CCCAAAGTGT(SEQ ID NO: 42)  Q  A  E   V  Q  L  V   E  S  G   G  G  V   V  Q  P  G  51 CCAGGCAGAA GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG  R  S  L   R  L  S   C  A  A  S   G  F  T   F  S  S  101GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGCY  G  M  H   W  V  R   Q  A  P   G  K  G  L   E  W  V  151TATGGCATGC ACTGGGTCCG CCAGGCTCCA GGCAAGGGGC TGGAGTGGGT A  I  I   W  Y  D  G   S  N  K   Y  Y  A   D  S  V  K  201GGCAATTATA TGGTATGATG GAAGTAATAA ATACTATGCG GACTCCGTGA  G  R  F   T  I  S   R  D  N  S   K  N  T   L  Y  L  251AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTGQ  M  N  S   L  R  A   E  D  T   A  V  Y  Y   C  A  R  301CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG L  W  F   G  D  L  D   A  F  D   I  W  G   Q  G  T  M  351GCTATGGTTC GGGGACTTAG ATGCTTTTGA TATCTGGGGC CAAGGGACAA  V  T  V   S  S  A   S  T  K  G   P  S  V   F  P  L  401TGGTCACCGT CTCCTCAGCC TCCACCAAGG GCCCATCGGT CTTCCCCCTGA  P  S  S   K  S  T   S  G  G   T  A  A  L   G  C  L  451GCACCCTCCT CCAAGAGCAC CTCTGGGGGC ACAGCGGCCC TGGGCTGCCT V  K  D   Y  F  P  E   P  V  T   V  S  W   N  S  G  A  501GGTCAAGGAC TACTTCCCCG AACCGGTGAC GGTGTCGTGG AACTCAGGCG  L  T  S   G  V  H   T  F  P  A   V  L  Q   S  S  G  551CCCTGACCAG CGGCGTGCAC ACCTTCCCGG CTGTCCTACA GTCCTCAGGAL  Y  S  L   S  S  V   V  T  V   P  S  S  S   L  G  T  601CTCTACTCCC TCAGCAGCGT CGTCACCGTG CCCTCCAGCA GCTTGGGCAC Q  T  Y   I  C  N  V   N  H  K   P  S  N   T  K  V  D  651CCAGACCTAC ATCTGCAACG TGAATCACAA GCCCAGCAAC ACCAAGGTGG  K  R  V   E  P  K   S  C  D  K   T  H  T   C  P  P  701ACAAGAGAGT TGAGCCCAAA TCTTGTGACA AAACTCACAC ATGCCCACCGC  P  A  P   E  L  L   G  G  P   S  V  F  L   F  P  P  751TGCCCAGCAC CTGAACTCCT GGGGGGACCG TCAGTCTTCC TCTTCCCCCC K  P  K   D  T  L  M   I  S  R   T  P  E   V  T  C  V  801AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG  V  V  D   V  S  H   E  D  P  E   V  K  F   N  W  Y  851TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTACV  D  G  V   E  V  H   N  A  K   T  K  P  R   E  E  Q  901GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA Y  N  S   T  Y  R  V   V  S  V   L  T  V   L  H  Q  D  951GTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG  W  L  N   G  K  E   Y  K  C  K   V  S  N   K  A  L 1001ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTCP  A  P  I   E  K  T   I  S  K   A  K  G  Q   P  R  E 1051CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA P  Q  V   Y  T  L  P   P  S  R   E  E  M   T  K  N  Q 1101ACCACAGGTG TACACCCTGC CCCCATCCCG GGAGGAGATG ACCAAGAACC  V  S  L   T  C  L   V  K  G  F   Y  P  S   D  I  A 1151AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCCV  E  W  E   S  N  G   Q  P  E   N  N  Y  K   T  T  P 1201GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC P  V  L   D  S  D  G   S  F  F   L  Y  S   K  L  T  V 1251TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTATAGC AAGCTCACCG  D  K  S   R  W  Q   Q  G  N  V   F  S  C   S  V  M 1301TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATGH  E  A  L   H  N  H   Y  T  Q   K  S  L  S   L  S  P 1351CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCCCC  G  K  * 1401GGGTAAATGA

The invention claimed is:
 1. A method for treating asthma, comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition comprising an antibody, or functionalfragment thereof, comprising H-CDR1, H-CDR2 and H-CDR3 selected from(i)-(ii): (i) SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, respectively;and (ii) SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10, respectively;and comprising L-CDR1, L-CDR2 and L-CDR3 selected from (iii)-(v): (iii)SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; (iv) SEQID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21, respectively; and (v) SEQID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 22, respectively; andcomprising a pharmaceutically acceptable carrier or excipient therefore.